Human chymase (EC.3.4.21.39), a chymotrypsin-like serine protease, is stored in mast cell secretory granules. Upon external stimulation, mast cells undergo degranulation, resulting in the release of human chymase, along with a wide variety of inflammation mediators, outside the cells. The released human chymase specifically recognizes aromatic amino acids contained in substrate proteins and peptides, such as phenylalanine and tyrosine, and cleaves the peptide bonds adjoining to the amino acids. A representative substrate for human chymase is angiotensin I (AngI). Human chymase cleaves AngI to produce angiotensin II (AngII), a vasoconstricting factor.
Mammalian chymases are phylogenetically classified under two subfamilies: α and β. Primates, including humans, express only one kind of chymase, which belongs to the α family. Meanwhile, rodents express both the α and β families of chymase. In mice, there are a plurality of kinds of chymases, of which mouse mast cell protease-4 (mMCP-4), which belongs to the β family, is considered to be most closely related to human chymase, judging from its substrate specificity and mode of expression in tissue. In hamsters, hamster chymase-1, also a member of the β family, corresponds to human chymase. Meanwhile, mMCP-5 and hamster chymase-2, which belong to the α family as with human chymase, possess elastase-like activity and differ from human chymase in terms of substrate specificity.
Chymase is profoundly associated with the activation of transforming growth factor β (TGF-β). TGF-β exists in a latent form (latent-TGF-β) in extracellular matrices around epithelial cells and endothelial cells, and is retained in extracellular matrices via large latent TGF-β binding protein (LTBP). TGF-β is released from extracellular matrices as required and activated, and the activated TGF-β is a cytokine of paramount importance to living organisms reportedly involved in cell proliferation and differentiation and tissue repair and regeneration after tissue injury. Collapse of its signal leads to the onset and progression of a wide variety of diseases. It is thought that in this process, chymase is involved in the release of latent TGF-β from extracellular matrices and the conversion of latent TGF-β to active TGF-β.
Chymase is known to be associated with a broad range of diseases, including fibrosis, cardiovascular diseases, inflammation, allergic diseases and organ adhesion. Fibrosis is an illness characterized by abnormal metabolism of extracellular substrates in the lung, heart, liver, kidney, skin and the like, resulting in excess deposition of connective tissue proteins. In pulmonary fibrosis, for example, connective tissue proteins such as collagen deposit in excess in the lung, resulting in hard shrinkage of pulmonary alveoli and ensuing respiratory distress. Lung fibrosis has been shown to result from pneumoconiosis, which is caused by exposure to a large amount of dust, drug-induced pneumonia, which is caused by use of drugs such as anticancer agents, allergic pneumonia, pulmonary tuberculosis, autoimmune diseases such as collagen disease, and the like. However, there are not a few cases in which the cause is unknown.
The mechanism of onset of fibrosis at the molecular level has not been elucidated well. Generally, in normal states, the proliferation and functions of fibroblasts are well controlled. In case of serious or persistent inflammation or injury, however, the tissue repair mechanism works in excess, resulting in abnormal proliferation of fibroblasts and overproduction of connective tissue proteins. TGF-β is known as a factor that causes these phenomena. As evidence suggestive of its involvement, it has been reported that administration of an anti-TGF-β neutralizing antibody to an animal model of fibrosis causes decreased collagen expression and significantly suppressed fibrosis. In patients with idiopathic pulmonary fibrosis, increased levels of TGF-β and elevated counts of chymase-positive mast cells are observed.
Meanwhile, association of chymase in fibrosis has been demonstrated by experiments using animal models. In a hamster model of bleomycin-induced pulmonary fibrosis, facilitated chymase activity, increased expression of collagen III mRNA, tissue fibrosis and other phenomena are significantly reduced by chymase inhibitors. The same effects have been observed for a mouse model of bleomycin-induced pulmonary fibrosis; administration of chymase inhibitors suppressed chymase activity and reduced hydroxyproline content.
With these features, chymase inhibitors can be used as prophylactic or therapeutic drugs for diseases related to chymase, such as fibrosis. Chymase inhibitors that have been developed include small molecular compounds such as TPC-806, SUN-13834, SUN-C8257, SUN-C8077, and JNJ-10311795 (Patent document 1).
In recent years, applications of RNA aptamers to therapeutic drugs, diagnostic reagents, and test reagents have been drawing attention; some RNA aptamers have already been in clinical study stage or in practical use. In December 2004, the world's first RNA aptamer drug, Macugen, was approved as a therapeutic drug for age-related macular degeneration in the US. An RNA aptamer refers to an RNA that binds specifically to a target molecule such as a protein, and can be prepared using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Patent documents 2-4). In the SELEX method, an RNA that binds specifically to a target molecule is selected from an RNA pool with about 1014 different nucleotide sequences. The RNA used has a random sequence of about 40 nucleotides, which is flanked by primer sequences. This RNA pool is allowed to mixed with a target molecule, and only the RNA that has bound to the target molecule is separated using a filter and the like. The RNA separated is amplified by RT-PCR, and this is used as a template for the next round. By repeating this operation about 10 times, an RNA aptamer that binds specifically to the target molecule can be acquired.
Aptamer drugs, like antibody drugs, can target extracellular proteins. With reference to many scientific papers and other reference materials in the public domain, aptamer drugs are judged to potentially surpass antibody drugs in some aspects. For example, aptamers often exhibit higher affinity and specificity for target molecules than do antibodies. Aptamers are unlikely to undergo immune elimination, and adverse reactions characteristic of antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), are reportedly unlikely to occur with the use of aptamers. From the viewpoint of drug delivery, aptamers are likely to migrate to tissues because of their molecular size of about one-tenth that of antibodies, enabling easier drug delivery to target sites. Because aptamers are produced by chemical synthesis, they permit site-selective chemical modifications, and enable cost reduction by mass-production. Other advantages of aptamers include long-term storage stability, heat resistance and solvent resistance. Meanwhile, the blood half-lives of aptamers are generally shorter than those of antibodies; however, this property is sometimes advantageous in view of toxicity. These facts lead to the conclusion that even when the same molecule is targeted, aptamer drugs potentially surpass antibody drugs.